Program Director/Principal Investigator (Hinck, Lindsay, E.): There is a fundamental gap in understanding the role of tissue specific stem/progenitor cells and their capacity for division and renewal. In tissues such as the breast, these cells undergo expansive growth and differentiation with every pregnancy. Yet, mechanisms regulating the generation of binucleated, milk-producing alveolar cells are scarcely understood. Until this knowledge gap is closed, we will be unable to support the substantial number of women who produce insufficient milk. The long-term goal of this research is to understand how mammals harness stem/progenitor cells to build a milk supply. Recently, it was discovered that a large fraction of cells become polyploid during mammary alveologenesis, a process required for milk production. This proposal's objective is to identify the signaling pathways regulating the generation of polyploid cells via endoreplication in response to DNA damage generated by replication stress, and determine the impact of these pathways on milk production. The central hypothesis is that ROBO-regulated NOTCH signaling governs the DNA damage differentiation response and endoreplication that occurs in response to alveolar progenitor cell expansion and differentiation. Our hypothesis is based on our own preliminary data that ROBOs differentially regulate alveologenesis and Notch signaling, and that, together, Robo/Notch signaling regulates the response to DNA damage, which we find occurring during alveologenesis. The rationale underlying this proposal is that the identification of these pathways will allow for interventions, pharmacological or dietary, that improve alveolar development and milk production. Guided by strong preliminary data, three hypotheses will be tested in three Aims: 1) ROBO1 promotes differentiation by restricting the activation of one or more NOTCH receptors in AVPs. 2) ROBO2 inhibits differentiation by activating the signaling of one or more Notch receptors either directly or by acting through ROBO1. 3) Replication stress, occurring during the expansion phase of pregnancy, is the source of DNA damage, triggering the DNA damage differentiation response and endoreplication that is governed by Robo/Notch signaling. The proposed research is significant because it will identify new methods for increasing milk production. The proposed research is innovative because we propose that harnessing stem/progenitor cells can enhance milk production. Previous studies have focused on the prolactin pathway and only yielded drugs with significant negative side effects. The proposed research will have a positive impact for women who produce insufficient milk and their children who do not reap the benefits of this ?liquid gold?. PHS 398 (Rev. 01/18 Approved Through 03/31/2020) Page Continuation Format Page